Many commonly used research methods, including cellular imaging via light and confocal microscopy, immunohistochemistry and fluorescent in situ hybridizations require the processing of thin and/or thick biological tissue sections over a glass slide or a glass-coverslip. If the tissue sections are well prepared and flat, and if the glass surface is clean, then the weak non-ionic forces, like hydrogen bonds and van der Waals forces, help the tissue sections to adhere to the glass surface.
However, often the tissue sections are not completely flat. Also, the slide surface may contain dust particles that can prevent good surface contact. Moreover, some tissue types do not even adhere to a slide or glass surface at all. Because of such problems, tissue sections are generally lost during tissue processing, which leads to wasted time and energy and rising costs.
For those sections that do adhere, tissue adhesion is generally not strong enough to withstand the mechanical stress of tissue processing. Examples of mechanical stress include, but are not limited to, long incubation periods in various chemicals, high temperatures and repeated washing in various solutions. Although these examples may be imperative to achieve successful processing of tissues, long processing times and repeated physical manipulation can dislodge the tissue.
The most commonly used method for preventing the dislodging of tissue sections is by coating the glass surface with various adhesives. Examples of adhesives include poly-L-Lysine (pLL), silane, glue (e.g., Elmer's glue), Mayere's egg albumin, chrome alum and chrome gelatin, silicon rubber, and starch paste. Another method involves creating a charged slide by using a known adhesive, such as pLL and silane.
There are a number of commercial products (e.g., adhesives and/or charged slides) that are available in the market that are aimed at improving the tissue retention. But, none of these completely solves the problem. Nonlimiting examples include Tissue-Tack Adhesive (Proscitech of Queensland, Australia), Biobond Tissue Section Adhesive (SPI Supplies of West Chester, Pa.), Superfrost Plus Gold Slides (Electron Microscopy Sciences of Hatfield, Pa.), Superfrost Plus Gold Slides (Erie Scientific Company of Portsmouth, N.H.), BD BioCoat Precoated Glass Coverslips (BD Biosciences of San Jose, Calif.), SUPERFROST PLUS—Adhesion (Electron Microscopy Sciences of Hatfield, Pa.), Poly-L-Lysine Coated and Silane Treated Microscope Slide (Electron Microscopy Sciences of Hatfield, Pa.), Excell Adhesion Slides (Electron Microscopy Sciences of Hatfield, Pa.), and Polysine Microscope Adhesion Slide (Electron Microscopy Sciences of Hatfield, Pa.).
It is even more challenging to mount thick tissue specimens. For example, a Drosophila cuticle has an uneven surface morphology and is more prone to curling up during dehydration. For some protocols, like immunohistochemistry, it is essential to dehydrate the cuticle well before mounting them for microscopy.
Conventionally, dehydration of the Drosophila larval cuticle is performed by mounting the filleted, fixed, and stained larval cuticle over a poly-L-Lysine (pLL) coated coverslip. Then, it is immersed sequentially into 30%, 50%, 90%, 100% and 100% ethanol solutions (˜5 minutes each). Finally, the cuticle cleared twice in xylene to remove the ethanol and make the sections optically transparent (˜10 minutes each).
Due to the thickness and uneven surface of the larval cuticle, it is very difficult to retain the tissues over the coverslip while maintaining section flatness during the dehydration process. A considerable percent of tissues usually dislodge from the glass surface due to insufficient adhesion to the coverslip. Once dislodged, it is difficult to recover the tissue for further analysis. Some tissues, even though not completely dislodged, curl in the edges, rendering them unfit for analysis. Further, the conventional method of dehydrating the cuticle requires considerable practice and lots of handling with care to minimize the tissue loss during processing. For example, someone with just a few months of experience in the protocol may lose about 50%-70% of total tissues processed due to poor tissue retention. Even a person with considerable experience may lose about 20%-30% tissues regularly. The percent of tissue retained during dehydration tends also to be strongly affected by the quality of pLL coating on the coverslips, which may suffer from batch-to-batch variations.
Furthermore, some methods require harsh treatment of sections (e.g., antigen retrieval), resulting in loss of tissue. Simply, these methods, along with the ones above, cannot guarantee complete tissue retention.
Consequently, what is needed is a device that allows all kinds of tissues to be retained over a flat surface (e.g., slide, coverslip, etc.) during the tissue processing for analysis. The device should also prevent the tissues from loosing its flat morphology. Furthermore, the device should be able to withstand the harsh conditions of tissue processing without affecting the quality of tissue processing.